Lhx9 is required for Hcrt neuron specification. (A-C) hcrt ISH at 24hpf shows that morpholino-mediated knockdown of lhx9 reduces the number of Hcrt neurons and the level of hcrt expression (A) compared with embryos injected with a control morpholino (B). (C) Quantification of Hcrt neurons per brain hemisphere at 24, 48 and 72hpf revealed that lhx9 morphants have ~40% fewer Hcrt neurons. Co-injecting a p53 morpholino did not affect this phenotype. Mean±s.e.m. is shown. At least 22 embryos were quantified for each condition. ***P<0.001 compared with control morpholino (one-way ANOVA followed by Bonferroni′s correction for multiple comparisons). (D-G) hcrt ISH at 24hpf shows that co-injection of Cas9 protein and ten lhx9 sgRNAs eliminates hcrt expression (D) compared with embryos injected with Cas9 alone (E). (F) Quantification of Hcrt neurons per brain hemisphere at 24hpf. Mean±s.e.m. is shown. ***P<0.001 compared with Cas9 alone (one-way ANOVA). (G) The percentage of brain hemispheres containing the indicated number of Hcrt neurons. n, number of brain hemispheres analyzed in F,G. Arrowheads indicate endogenous Hcrt neuron region. Scale bars: 50µm.